Epidemiological studies have suggested an association between pesticide exposure and Parkinson's disease. In this study, we examined the neurotoxicity of an organochlorine pesticide, heptachlor, in vitro and in vivo. In cultured SH-SY5Y cells, heptachlor induced mitochondria-mediated apoptosis. When injected into mice intraperitoneally on a subchronic schedule, heptachlor induced selective loss of dopaminergic neurons in the substantia nigra pars compacta. In addition, the heptachlor injection induced gliosis of microglia and astrocytes selectively in the ventral midbrain area. When the general locomotor activities were monitored by open field test, the heptachlor injection did not induce any gross motor dysfunction. However, the compound induced Parkinsonism-like movement deficits when assessed by a gait and a pole test. These results suggest that heptachlor can induce Parkinson's disease-related neurotoxicities in vivo.
Animals ; *Apoptosis ; Astrocytes/drug effects/pathology ; Cell Line, Tumor ; Cells, Cultured ; Dopaminergic Neurons/*drug effects/pathology ; Gait ; Heptachlor/*toxicity ; Humans ; *Locomotion ; Mice ; Neurotoxicity Syndromes/etiology/physiopathology ; Parkinsonian Disorders/chemically induced ; Pesticides/*toxicity ; Substantia Nigra/*drug effects/pathology/physiopathology

To investigate vincristine-induced dopaminergic neurons toxicity and mechanism, and explore the molecular target to reduce the toxicity, zebrafish was chosen as a model animal, based on RT-PCR, Western blotting, whole mount in situ immunofluorescence and other technical means. The results showed that the transcription levels of tyrosine hydroxylase gene and dopamine transporter protein gene were inhibited. Furthermore, the number of dopaminergic neurons was decreased by vincristine. Autophagy was suppressed and beclin1 gene expression was inhibited in a dose-dependent manner by vincristine in larval zebrafish. Up-regulated beclin1 partly reduced vincristine-induced neurotoxicity, and down-regulated beclin1 increased toxicity. Beclin1 plays an important role in vincristine-induced dopaminergic neurons toxicity.
Animals ; Apoptosis Regulatory Proteins ; metabolism ; Autophagy ; Dopaminergic Neurons ; drug effects ; pathology ; Dose-Response Relationship, Drug ; Down-Regulation ; Gene Expression Regulation ; drug effects ; Larva ; drug effects ; Tyrosine 3-Monooxygenase ; metabolism ; Vincristine ; adverse effects ; Zebrafish ; Zebrafish Proteins ; metabolism

MicroRNAs (miRNAs) are a class of noncoding RNAs that regulates target gene expression at posttranscriptional level, leading to further biological functions. We have demonstrated that microvesicles (MVs) can deliver miRNAs into target cells as a novel way of intercellular communication. It is reported that in central nervous system, glial cells release MVs, which modulate neuronal function in normal condition. To elucidate the potential role of glial MVs in disease, we evaluated the effects of secreted astrocytic MVs on stress condition. Our results demonstrated that after Lipopolysaccharide (LPS) stimulation, astrocytes released shedding vesicles (SVs) that enhanced vulnerability of dopaminergic neurons to neurotoxin. Further investigation showed that increased astrocytic miR-34a in SVs was involved in this progress via targeting anti-apoptotic protein Bcl-2 in dopaminergic neurons. We also found that inhibition of astrocytic miR-34a after LPS stimulation can postpone dopaminergic neuron loss under neurotoxin stress. These data revealed a novel mechanism underlying astrocyte-neuron interaction in disease.
Animals ; Astrocytes ; cytology ; drug effects ; metabolism ; Cell Line, Tumor ; Cell Survival ; drug effects ; Cell-Derived Microparticles ; metabolism ; Disease Models, Animal ; Dopaminergic Neurons ; drug effects ; pathology ; Down-Regulation ; drug effects ; Humans ; Lipopolysaccharides ; pharmacology ; MicroRNAs ; metabolism ; Neurotoxins ; toxicity ; Oxidopamine ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Rats ; Stress, Physiological ; drug effects

OBJECTIVETo discuss the protective effect of alkaloids from Piper longum (PLA) in rat dopaminergic neuron injury of 6-OHDA-induced Parkinson's disease and its possible mechanism.

METHODThe rat PD model was established by injecting 6-OHDA into the unilateral striatum with a brain solid positioner. The PD rats were divided into the PLA group (50 mg x kg(-1) x d(-1)), the madorpa group (50 mg x kg(-1) x d(-1)) and the model group, with 15 rats in each group. All of the rats were orally given drugs once a day for 6 weeks. Meanwhile, other 15 rats were randomly selected as the sham operation group, and only injected with normal saline in the unilateral striatum. The behavioral changes were observed with the apomorphine (APO)-induced rotation and rotary rod tests. The number of tyrosine hydroxylase (TH)-positive cells in rat substantia nigra and the density of TH-positive fibers in striatum were detected by tyrosine hydroxylase immunohistochemistry. The content of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), glutathione (GSH), catalase (CAT), malondialdehyde (MDA), nitric oxide (NO) and nitric oxide synthase (NOS) in rat substantia nigra and striatum were measured by the spectrophotometric method.

RESULTAfter being induced by APO, PD rats showed obvious rotation behaviors, with decreased time stay on rotary rod and significant reduction in the number of TH-positive cells in sustantia nigra and the density of TH-positive fibers in striatum. The activities of SOD, GSH-Px, CAT, the content of GSH and the total antioxidant capacity significantly decreased, whereas the activities of NOS and the content of MDA, NO significantly increased. PLA could significantly improve the behavioral abnormality of PD rats and increase the number of TH-positive cells in sustantia nigra and the density of TH-positive fibers in striatum. It could up-regulate the activities of SOD, GSH-Px, CAT, the content of GSH and the total antioxidant capacity, and decrease the content of NOS and the content of MDA, NO.

CONCLUSIONAlkaloids from P. longum shows the protective effect in substantia nigra cells of 6-OHDA-induced PD model rats. Its mechanism may be related with their antioxidant activity.

Administration, Oral ; Alkaloids ; administration & dosage ; pharmacology ; Animals ; Apomorphine ; pharmacology ; Catalase ; metabolism ; Dopamine Agonists ; pharmacology ; Dopaminergic Neurons ; drug effects ; metabolism ; pathology ; Glutathione ; metabolism ; Glutathione Peroxidase ; metabolism ; Male ; Malondialdehyde ; metabolism ; Motor Activity ; drug effects ; Neostriatum ; drug effects ; metabolism ; Nitric Oxide ; metabolism ; Nitric Oxide Synthase ; metabolism ; Oxidopamine ; Parkinson Disease, Secondary ; chemically induced ; physiopathology ; prevention & control ; Piper ; chemistry ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Substantia Nigra ; drug effects ; metabolism ; Superoxide Dismutase ; metabolism ; Tyrosine 3-Monooxygenase ; metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.
Animals ; Brain/pathology ; *Cell Differentiation/drug effects ; Cells, Cultured ; Culture Media/chemistry/pharmacology ; Dental Pulp/*cytology ; Dopaminergic Neurons/*cytology/*metabolism/pathology ; Enzyme-Linked Immunosorbent Assay ; Glial Fibrillary Acidic Protein/genetics/metabolism ; Humans ; Mice ; Mice, Inbred ICR ; Myelin Basic Protein/genetics/metabolism ; Real-Time Polymerase Chain Reaction ; Stage-Specific Embryonic Antigens/genetics/metabolism ; Stem Cells/*cytology/*metabolism/pathology ; Tubulin/genetics/metabolism ; Tyrosine 3-Monooxygenase/analysis/genetics/metabolism